MECHANISM OF PROTEIN TRANSLOCATION ACROSS MEMBRANES

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The primary objective of the proposed research is to understand the molecular mechanism by which certain classes of proteins are selectively targeted to and translocated across the endoplasmic reticulum (ER) membrane in mammalian cells. We will continue to explore biochemical, molecular cloning and immunological approaches to extend our knowledge of the structure and function of the known components of the protein translocation machine (namely the signal recognition particle (SRP), the SRP receptor and signal peptidase (SPase), as well as to identify and characterize further components that are involved in the process. Specifically, i) We will map the contact sites between sites between SRP and the ribosome and between SRP and SRP receptor. ii) We will purify and characterize soluble proteins and peripheral membrane proteins that, in addition to SRP and SRP receptor, are required for protein translocation, or that stimulate the process. Their role in protein translocation will be elucidated through the use of detailed in vitro assay. iii) We will further characterize and dissect already identified membrane components (such as SRP receptor and the SPase complex) with respect to their functional and structural domains. iv) We will use these components in affinity and crosslinking approaches to identify interacting proteins and attempt to define their functional role with immunological reagents. v) We will employ reconstitution of translocation activity into artificial lipid vesicles from detergent extracts of microsomal vesicles as an assay to fractionate and purify membrane components. vi) We will investigate the function of known or putative components of the translocation machine by using antibodies against these proteins to deplete the detergent extract prior to reconstitution. Ultimately, we hope that we will identify the cellular constituents of the protein translocation machine that are essential, as well as those that are modulatory, and gain a detailed understanding of the mechanism of protein translocation and the role which individual molecules play in this fundamentally important process. The proposed research is clearly of a most basic nature and there is no doubt that it will be of profound significance for an understanding of physiology and pathology at the cellular and molecular level.